CN104650210A - Method of producing recombinant TAT-HOXB4H protein for use as a stimulant of hematopoiesis in vivo - Google Patents
Method of producing recombinant TAT-HOXB4H protein for use as a stimulant of hematopoiesis in vivo Download PDFInfo
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- CN104650210A CN104650210A CN201410854099.1A CN201410854099A CN104650210A CN 104650210 A CN104650210 A CN 104650210A CN 201410854099 A CN201410854099 A CN 201410854099A CN 104650210 A CN104650210 A CN 104650210A
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Abstract
The invention discloses a method of producing recombinant TAT-HOXB4H protein for use as a stimulant of hematopoiesis in vivo, the recombinant TAT-HOXB4H protein, when administered to a subject in need thereof, increase number of HSCs in both the bone marrow and peripheral blood in vivo and relates to a C-terminal histidine tagged TAT-HOXB4 fusion protein (TAT-HOXB4H). The recombinant TAT-HOXB4H protein solves the problem that a recombinant protein obtained by purification of nutrient solution is low in quality and poor in stability. Improvement on subsequent purification and storage of buffering liquid of the recombinant TAT-HOXB4H protein is made, so that the recombinant TAT-HOXB4H protein is applicable to prepare medical combination directly administered in vivo. The absolute number of HSCs in the bone marrow of a subject is increased, thereby enhancing the mobilization of HSCs to the peripheral blood of the subject.
Description
The invention and created name that the application is the applying date is on May 27th, 2008, application number is 200810111350.X is the divisional application of the Chinese patent application of " TAT-HOXB4H recombinant protein and medical constituent thereof as hematopoiesis irritant ".
Technical field
The present invention relates to a kind of C-and hold the HOXB4 recombinant protein marked containing histidine, in particular to the recombinant protein method of a kind of C end containing six histidine residue (residue), the composition of recombinant protein prepared by described method can be applicable to for the preparation of in vivo offeing medicine to promote hemopoietic stem cell by bone marrow mobilization to the medicine of periphery blood.
Background technology
Develop flourishing today at regenerative medicine, various about find organ specific stem cells or can the research of oneself more raw (self-renewing) cell continuous.Described can the most studied in the more raw cell of oneself be hemopoietic stem cell because from cancer, disease of metabolism to immunological incompetence, hemopoietic stem cell is all good therapeutic choice.
" hematopoiesis " refers to the process that blood cell generates, and replaces red blood corpuscle and white cell by being arranged in the division of the hemopoietic stem cell of marrow.Hemopoietic stem cell (HSCs) is that a group has self-renewal capacity and the cell being divided into all blood cells or immunocyte ability.But the gene machine controlling hemopoietic stem cell self and differentiation turns most and still belongs to unknown.
At present, from Adult Human Bone Marrow, the mankind hemopoietic stem cell mobilizing rear perimeter edge blood and Umbilical Cord Blood Transplantation, be used for the treatment of leukemia (leukemia and lymphoma) clinically, and for helping immunity system to be restored by the high dose chemotherapy of non-leukemia.But the effective hemopoietic stem cell transplanting a large amount of different sources of needs, and may need to carry out stem cell proliferation (expansion).
Hemopoietic stem cell can from marrow, periphery blood and Cord blood.Take out medullary cell to need to carry out performing the operation and quite painful step, thus become poor means.Use periphery blood cell also defectiveness because be difficult to by hematopoietic function impaired fall ill or patients undergoing chemotherapy obtains suitable with enough hemopoietic stem cells.Cord blood relatively easily obtains and the quality of hemopoietic stem cell is also better, but the hemopoietic stem cell quantity that this method can obtain is still limited.The cell quantity at every turn obtained is enough for child, but just possibility is not enough will to be used for adult.For solving the problem, the more raw process of stem cell oneself just must be disturbed to accelerate the external hyperplasia of hemopoietic stem cell.
Recently evidence point out transcriptional regulator regulation and control stem cell gene expression, play an important role (Orkin, S.H.Nat.Rev.Genet 1,57-64,2000) with the growth course of stem cell.Transcriptional regulator by with the combination of target gene whether or concentration own forms complicated cell physiological regulatory mechanism.A group is called that the transcription factor of DNA binding homebox (HOX) is found in fetal development and plays an important role, and also play key player (Buske, C.et.al.J.Hematol.71,301-308,2000) at discovered in recent years HOX transcription factor family in hemopoietic stem cell growth.Lid savart lattice (Guy Sauvageau) doctor of Montreal university (University of Montreal) once inquired into the phenomenon of the HOX transcription factor regulation and control hemopoietic stem cell renewal of hemopoietic stem cell in marrow, its research display coordination sequence gene (homeobox gene) HOXB4 is very important for the regulation and control of hemopoietic stem cell self, can maintain the cluster size of hemopoietic stem cell in marrow.First proving hox gene can show at blood cell, is utilize the cell strain (cell line) of the mankind and mouse and confirm.Some hox gene has in different cellular fories and obviously shows widely, some hox gene then only activation find expression in specific cells.Such as: eight members that the HOXB of the mankind goes here and there in group can show at the beginning of red blood cell is grown, some HOXB gene comprises HOXB4 and HOXB7 and also can show in T cell and B cell.The people such as Sauvageau confirms to have nine HOXA genes, eight HOXB genes and four HOXC genes can at CD34
+performance in medullary cell, wherein again with HOXB2, HOXB9 and HOXA10 at CD34
+cell populations in the performance of red blood corpuscle initiating cell at most.In addition, experimental result also detects at CD34
-cell mass in, do not have hox gene to show.Therefore, " HOXB4 " albumen has the most often been used to the effective stimulus agent as in vitro hemopoietic stem cell (HSC) hyperplasia.Recently confirm the mankind, " HOXB4 " gene can virus or the next effective hyperplasia hemopoietic stem cell of recombinant protein form.TAT-HOXB4H recombinant protein, at laboratory-grade, be used to hyperplasia stem cell and do not had the risk of retrovirus insertion or the risk (see Krosl, J.et al., Nature Medicine 9,1428-1432,2003) with marrow stromal cell Dual culture.Therefore, " HOXB4 " albumen has been usually used in the stimulant as promoting in vitro hemopoietic stem cell hyperplasia.
Existing evidence points out that the HOXB4 of external source can be transported in cell after the N end of HOXB4 adds a TAT protein sequence recently.This TAT sequence system can guide HOXB4 by extracellular to intracellular transport.Once enter tenuigenin, HOXB4 can by escorting albumen (Chaperon) HSP90 and the folding configuration that becomes it original again.TAT-HOXB4 can promote that hemopoietic stem cell hyperplasia reaches 2-6 doubly (people such as Amsellem, S., NatureMedicine 9,1423-1427,2003; And the people such as Krosl, J., Nature Medicine 9,1428-1432,2003).But it is on the low side that this TAT-HOXB4 recombinant protein reclaims productive rate from the purifying of escherichia coli host, and mostly be soluble form.
In order to increase the productive rate of this TAT-HOXB4 recombinant protein, developed C-and held the TAT-HOXB4H recombinant protein manufacture method separately having one section 6 histidine residue (His-6) and mark, its productive rate exceeds 3-5 doubly compared with the purification efficiency of urporotein.This manufacture method is described in detail in PCT/CN2006/000646.
Above-mentioned TAT-HOXB4H recombinant protein can be used for human peripheral's blood or cord blood stem cell hyperplasia, and the stem cell of institute's hyperplasia still possesses its versatility (pluripotency).In addition, the stem cell of above-mentioned TAT-HOXB4H recombinant protein process is added in non-obese patients with type Ⅰ DM merge severe combined immunodeficient mouse (NOD/SCID) marrow in after, human leukocyte can be found, it can thus be appreciated that mouse immune and hematopoietic function successful reconstitution in its peripheral leukocytes.
But, TAT-HOXB4H recombinant protein is never used to the stimulant as hematopoiesis in body, particularly, never be used to promote that hematopoietic function is rebuild, amplification, marrow lives again (re-population) and increase the number of peripheral circulation stem cell, particularly after chemotherapy or radiation cure.The people (2003) such as the people such as Krosl (2003) and Amsellem cannot obtain a large amount of high purity needed for amplifying candidate stem cell clinical study and the HOXB4 protein highly stabilized.
The TAT-HOXB4H recombinant protein total amount using prior art to be obtained by one liter of nutrient solution purifying is about 1-2 milligram, and productive rate is lower.Use the pTAT-HA-HOXB4 plastid of art methods performance TAT-HOXB4H protein, that lid savart lattice (GuySauvageau) doctor of Canadian Montreal university (University of Montreal) given, the people such as Krosl (2003) once reported, at serum free culture system after 4 hours, the TAT-HOXB4H protein of its purifying can lose major part.
Summary of the invention
The present invention is based on aftermentioned result of study: after TAT-HOXB4H recombinant protein gives a user in need, it can increase the number of the hemopoietic stem cell in marrow and in periphery blood.
One aspect of the present invention provides a kind of TAT-HOXB4H protein production methods.The method comprises:
A () provides a host cell, it comprises the carrier that has above-mentioned protein coding;
B () shows this protein in this host cell;
C () collects an impure solution of this performance protein;
(d) with the following step by this protein of this solution purification: this solution is passed into a HisTrap tubing string by (i); (ii) this HisTrap tubing string is rinsed; (iii) this partially purified protein is molten from going out by this HisTrap tubing string, and form the protein soln of a part of purifying; (iv) this partially purified protein soln is passed into a MonoSP tubing string; V () rinses this MonoSP tubing string; (vi) by the protein of purifying, in denatured form, molten from going out by this MonoSP tubing string;
E () utilizes hydrophobic compound by the molten denatured protein from going out with the following step folding again: this molten denatured protein from going out and a hydrophobic compound solution are mixed to form a protein and hydrophobic compound solution by (i); (ii) this protein and hydrophobic compound solution are desalted and a protein and hydrophobic compound desalt solution; (iii) utilize ultra-filtration processing procedure to be desalted in solution by this protein and hydrophobic compound by this hydrophobic compound to remove.
The present invention provides on the other hand and a kind ofly promotes hemopoietic stem cell by bone marrow mobilization to the method for periphery blood.The method comprises:
A) the TAT-HOXB4H recombinant protein manufactured with aforesaid method of user one significant quantity in need is given;
B) allow this TAT-HOXB4H recombinant protein to increase the absolute number of hemopoietic stem cell in this user's marrow, promote that hematopoietic stem cell mobilization is to the periphery blood of this user thus.
Further aspect of the present invention is to providing a kind of method of time of recovery in order to improve patients with hematopoietic stem cells transplantation, radiation cure patient or patients undergoing chemotherapy.The method comprises:
A) the TAT-HOXB4H recombinant protein manufactured with aforesaid method of user one significant quantity in need is given;
B) this TAT-HOXB4H recombinant protein is allowed to increase the absolute number of hemopoietic stem cell in this user's marrow.
Further aspect of the present invention provides a kind of in order to promote hemopoietic stem cell by the bone marrow mobilization of a user in need to the medical constituent of periphery blood.Medical constituent of the present invention comprises the TAT-HOXB4H recombinant protein manufactured with aforesaid method of a significant quantity, and it is enough to the absolute number increasing hemopoietic stem cell in this user's marrow, promotes that hematopoietic stem cell mobilization is to the periphery blood of this user thus.
Medical constituent of the present invention can carry out the patient of autologous hematopoietic stem cell transplantation, in order to improve the time of recovery after its hematopoietic stem cell transplantation.
Medical constituent of the present invention, can be used as the surrogate of particle glomus cell somatomedin (G-CSF), gives the insensitive patient of G-CSF, in order to mobilizing hematopoietic stem cells to periphery blood.
Medical constituent of the present invention can give hemopoietic stem cell contributor, can obtain enough hemopoietic stem cells for transplanting thus, and must do not obtained by its marrow by the Peripheral blood of contributor in less invasive mode.
Further aspect of the present invention provides the congenital hemopoietic stem cell for the treatment of and lacks and the disease that causes, and it is by systemic applications mode, gives the TAT-HOXB4H recombinant protein manufactured with aforesaid method or its medical constituent of above-mentioned Disease one significant quantity.The TAT-HOXB4H recombinant protein given can increase the absolute number of hemopoietic stem cell in this user's marrow.
Further aspect of the present invention provides a kind of method of time of recovery in order to improve patients with hematopoietic stem cells transplantation, it is by systemic applications mode, gives the TAT-HOXB4H recombinant protein manufactured with aforesaid method or its medical constituent of user one significant quantity in need.
Further aspect of the present invention provides a kind of method that hemopoietic stem cell in order to improve radiation cure patient or patients undergoing chemotherapy is replied, it is by systemic applications mode, gives the TAT-HOXB4H recombinant protein manufactured with aforesaid method or its medical constituent of user one significant quantity in need.
Accompanying drawing explanation
Fig. 1 is that in the present invention, hemopoietic stem cell (HSC) mobilizes the schematic diagram to Peripheral blood (PB) from amplification in body;
Fig. 2 is that the choosing of pTAT-HOXB4H in the present invention is grown and the schematic diagram constructed in the pET21b plastid revised;
Fig. 3 is the DNA sequence dna figure of pTAT-HOXB4H in the present invention; (pTAT-HOXB4HN-end and C-hold six extra histidine residue to add bottom line and indicate, and put on TAT)
Fig. 4 is the protein sequence figure of pTAT-HOXB4H in the present invention;
Fig. 5 proves TAT-HOXB4H protein purification with 10%SDS-polyacrylamide gel (1.5mm) in the present invention, and it is with the protein sequence of coomassie blue staining analysis diagram pTAT-HOXB4H;
Wherein: M: molecular weight marker (M), 0.3 μ g;
Lane 1: from the cell lysates of not inducing BL21 (DE3) pLysS TAT-HOXB4H protein to show cell, 1 μ g protein;
Lane 2: after carrying out self-induction, BL21 (DE3) pLysS TAT-HOXB4H protein shows the cell lysates of cell, 1 μ g protein;
Lane 3: the TAT-HOXB4H of purifying, 0.7 μ g protein;
Lane 4: the TAT-HOXB4 (0.2 μ g protein) of purifying; In order to show the pTAT-HA-HOXB4 plastid of TAT-HOXB4 protein (lane 5), be that lid savart lattice (Guy Sauvageau) doctor of Canadian Montreal university (University of Montreal) given.
Lane 3 and lane 4 is the stream parts (fraction) of being collected by MonoSP tubing string adding same volume.
Fig. 6 is with the stability figure of TAT-HOXB4H protein after SDS-polyacrylamide gel analysis purifying in the present invention; (it is stored in PBS, 4 DEG C, 0 hour (A) and 16 hours (B), wherein: M represents molecular weight marker, 0 representative 4 DEG C 0 hour, 16 representatives 4 DEG C 16 hours)
Fig. 7 be in the present invention with SDS-polyacrylamide gel and coomassie staining analysis TAT-HOXB4H protein storage at-4 DEG C and-20 DEG C, the stability figure of PBS and IMDM damping fluid; (arrow instruction TAT-HOXB4H protein band)
Fig. 8 A be in the present invention with flow cytometry analysis G-CSF for CD34 in mouse bone marrow cells
+the effect of stimulation figure of stem cell population;
Fig. 8 B be in the present invention with flow cytometry analysis PBS for CD34 in mouse bone marrow cells
+the effect of stimulation figure of stem cell population;
Fig. 8 C be in the present invention with flow cytometry analysis TAT-HOXB4H protein for CD34 in mouse bone marrow cells
+the effect of stimulation figure of stem cell population;
Fig. 9 A be in the present invention with flow cytometry analysis G-CSF for CD34 in rhesus monkey marrow
+the effect of stimulation figure of stem cell population; .
With flow cytometry analysis TAT-HOXB4H protein, Fig. 9 B adds that G-CSF is for CD34 in rhesus monkey marrow in the present invention
+the effect of stimulation figure of stem cell population;
Fig. 9 C be in the present invention with flow cytometry analysis TAT-HOXB4H protein for CD34 in rhesus monkey marrow
+the effect of stimulation figure of stem cell population;
Fig. 9 D be in the present invention with flow cytometry analysis PBS for CD34 in rhesus monkey marrow
+the effect of stimulation figure of stem cell population;
Figure 10 is that in the present invention, TAT-HOXB4H protein replys effect diagram for the hematopoiesis of NOD-SCID mouse;
Figure 11 is that in the present invention, TAT-HOXB4H protein replys effect diagram for the hematopoiesis accepting Balb/c mouse after Cisplatin chemotherapy.
Embodiment
Below in conjunction with the drawings and specific embodiments, embodiments of the invention are described in further detail.
I.TAT-HOXB4H protein
The TAT-HOXB4H recombinant protein total amount that the present invention is obtained by one liter of nutrient solution purifying is about 6-10 milligram, and TAT-HOXB4H method of purifying protein of the present invention has in body the productive rate giving the required lifting of protein.TAT-HOXB4H protein of the present invention, even if after serum free culture system 4 week, still shows obviously preferably stability, TAT-HOXB4H protein is used for the key of clinical study herein.
The manufacture method of TAT-HOXB4H protein provided by the present invention, has the advantage that can improve productive rate and stability, makes above-mentioned protein can be used for vivo medicine-feeding.This TAT-HOXB4H protein is for comprising the structure body (construct) of three gene fragments (element) (TAT, HOXB4 and histidine mark).HOXB4 is a member of transcriptional regulator HOX family, and it can promote hemopoietic stem cell hyperplasia.TAT makes HOXB4 part be transported in nucleus.Histidine mark can make restructuring show the initial purification gain in yield in source, and manufacture method of the present invention can increase protein yields further.PTAT-HOXB4H constructs as shown in Figure 2, and DNA sequence dna system as shown in Figure 3.TAT-HOXB4H recombinant protein means the TAT-HOXB4 fused protein of C end containing six histidine residue (residue) mark (see Fig. 4).
Except as otherwise noted, the aminoacid sequence (or claiming " primary structure " or " primary series ") of protein is to carboxyl terminal by aminoterminal.Abiotic system (such as using solid state synthesis person), the primary structure of protein (comprising two sulphur (halfcystine) key position) can be custom by user.
" delete (deletion) " and refer to the change that amino acid (or Nucleotide) sequence causes owing to lacking one or more amino-acid residue (or Nucleotide)." insert (insertion) or increase " and referring to that the change of amino acid (or Nucleotide) sequence causes at a part or its manifestation, compared to a canonical sequence (such as naturally there is the sequence that molecule finds), increase one or more amino-acid residue (or Nucleotide)." replacement " refers to that one or more amino acid (or Nucleotide) is replaced by different amino acid (or Nucleotide).
Also be a part of the present invention with the sequence of sequence similarity of the present invention or homology (such as sequence have 70% identical).In certain embodiments, sequence is mutually unison can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.In addition, essence identical be also present in when nucleic acid fragment can stock complementary with it hybridization (such as under highly strict condition) time.Nucleic acid can appear at full cell, cytolysis thing or be half purifying or purified form.
Between two sequences, the method for calculation of " homology " or " sequence thereto " (both are commutative use herein) are as follows.First sequence is alignd (such as can add that breach (gap) compares with the most applicable in the first or second amino acid (or nucleic acid) sequence, and can remove the fragment of not homology when comparing) in the most applicable mode compared.In a preferred embodiment of the invention, the length that canonical sequence compares for aliging can be at least 30%, better at least 40%, better at least 50%, better at least 60%, better at least 70%, 80%, 90%, 100% of canonical sequence length.Then the amino-acid residue (or Nucleotide) on correspondence position is compared.When First ray and identical amino-acid residue (or the Nucleotide) of the second sequence opposite position, then identical in this position (referring to be equal to amino acid (or nucleic acid) " homology " this amino acid (or nucleic acid) " identical ").Mutually unison per-cent between two sequences refers to the function of the number owning same position between two sequences together, and it need consider to align the number of breach and the length of each breach added in order to optimizing.
Gene comparision between two sequences and same percentage can utilize a mathematical algorithm to reach.Preferably implement in profit at one of the present invention, same percentage between two aminoacid sequences can utilize Needleman and Wunsch (1970) (J.Mol.Biol.48:444-453) algorithm being incorporated to GCG software package (http://www.gcg.com) GAP program to determine, use Blossum 62matrix or PAM250matrix and 16,14,12,10,8, the gap weight and 1 of 6 or 4,2,3,4, the length weight of 5 or 6.Preferably implement in profit at of the present invention another, same percentage between two aminoacid sequences can utilize GCG software package (http://www.gcg.com) to determine, use NWSgapdna.CMP matrix and 40,50,60, the gap weight and 1 of 70 or 80,2,3,4, the length weight of 5 or 6.One group of preferred parameter group (when which parameter uncertain can in order to determine that whether a part is the sequence of the present invention's identical or homology time, this group parameter can be used) be use the Gap Penalty (penalty) of Blossum 62scoring matrix and 12, the gap extension penalty of 4 and framework displacement (frame shift) Gap Penalty of 5.Same percentage between two amino acid (or Nucleotide) sequence also can utilize E.Meyersand W.Miller ((1989) CABIOS, 4:11-17) algorithm of the ALIGN program that has been incorporated to (2.0 editions) to determine, uses the Gap Length Penalty of PAM120weight residuetable and 12 and the Gap Penalty of 4.
II.TAT-HOXB4H recombinant protein manufacture method
A. choosing is grown and performance
Grow the system with performance protein for choosing in various host cell, be widely known by the people in the art.Be applicable to the cell manufacturing protein, such as: Fernandez et al. (1999) Gene ExpressionSystems.In brief, the host cell be applicable to comprises mammalian cell, insect cell, vegetable cell, yeast cell or prokaryotic cell prokaryocyte (such as E.coli).For the mammalian cell of heterologous protein performance in this area, comprise lymphocyte strain (such as NSO), HEK293cells, Chinese hamster ovary cell (CHO), COS cells, HeLa cells, baby hamster kidney cell, ovocyte and the cell from Transgenic animals, such as: mammary epithelial cell.Suitable carrier can be built into containing suitable adjustment sequence, comprises promoter sequence, gene termination sequence, poly-adenosine sequence, enhancer sequence, marker gene and other sequence.Carrier can contain plastid or viral backbone.Details is see Sambrook et al.:Molecular Cloning:A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press (1989).Many carrier correlation techniques, comprise operation, preparation, form sudden change, sequence understands and DNA transfection (transfection) is described in Current Protocols in MolecularBiology, Second Edition, Ausubel et al.eds., John Wiley & Sons (1992).
Present invention also offers a kind of method Nucleotide being sent into host cell.For prokaryotic cell prokaryocyte, the rotaring dyeing technology be applicable to comprises calcium phosphate, DEAE-Dextran, electroporation, the transfection of lipophore intermediary and utilizes retrovirus or other virus, such as: vaccinia virus (vaccinia) or baculovirus (baculovirus).For bacterial cell, applicable technology comprises calcium chloride transformation (transformation), electroporation and utilizes the transfection of lipophore intermediary and utilize phage transfection.DNA then can select the cell containing Nucleotide with system of selection (such as drug resistance) after sending into.
B. purifying with at folding again
TAT-HOXB4H protein can utilize any known suitable means in this area isolated by recombinant host cell.Such as: when protein can secreted time, it can be separated by cell conditioned medium liquid; Or protein can be separated by cell lysates.
TAT-HOXB4H protein can utilize purification by chromatography, and it comprises: cell lysates or cell conditioned medium liquid (if protein can be secreted) are passed into a HisTrap tubing string by (a); B () rinses this HisTrap tubing string with a buffered soln; C () is molten from going out by this HisTrap tubing string by this partially purified protein; D the partially purified protein received by HisTrap tubing string is passed into a MonoSP tubing string by (); E () rinses this MonoSP tubing string with a buffered soln; F () is molten from going out by MonoSP tubing string by the protein of purifying.
Cell lysates or cell conditioned medium liquid (if protein can be secreted) can make it clarify in centrifugal 30 minutes with 20,000x g at 4 DEG C.Supernatant liquor is adjusted to 10mM imidazo and adds HisTrap chelating tubing string (Amersham Pharmacia).With 8M urea, 20mM HEPES, 0.5mM DTT, 100mM NaCl pH8.0 buffered soln and 10mM imidazoles washover pipe, to remove unbinding protein.Partially purified protein can utilize the imidazoles of high density and salt by this HisTrap tubing string molten from.
Be further purified and refer to the partially purified protein received by HisTrap tubing string to pass into MonoSP tubing string (Amersham Pharmacia).With 4M urea, 20mM HEPES, 50mM NaCl pH6.5 buffered soln one buffered soln washover pipe, to remove unbinding protein.In conjunction with TAT-HOXB4H can utilize the salt of high density molten from.The TAT-HOXB4H protein system of this purification step gained is denatured form.
Then, utilize hydrophobic compound by by HisTrap tubing string molten from the TAT-HOXB4H protein of sex change with the following step folding again: this molten denatured protein from going out and a hydrophobic compound solution are mixed to form a protein and hydrophobic compound solution by (i); (ii) this protein and hydrophobic compound solution are desalted and a protein and hydrophobic compound desalt solution; (iii) utilize ultra-filtration processing procedure to be desalted in solution by this protein and hydrophobic compound by this hydrophobic compound to remove.
In the present invention, " hydrophobic compound " refers to and anyly can desalt in step in sex change, and protected protein matter makes it not produce the long-pending hydrophobic compound in insoluble Shen.Be applicable to hydrophobic compound system of the present invention and be described in Oganesyan et al., Pharmagenomics (2004) 71,22-26.Suitable hydrophobic compound comprises Triton X-100, tween-20 or many benzene ring compound.This ultra-filtration processing procedure or buffered soln conversion can be undertaken by protein concentration tubing string (centricon tube) or albumen ultra-filtration membrane (stir cell).This ultra-filtration processing procedure or buffered soln conversion condition opsin matter kind and determine.
In one embodiment of the invention, remove by buffered soln conversion (at every turn with 1000-2500x g centrifugal 10 minutes) of 5-10 time in the hydrophobic compound system of desalting in solution containing HOXB4H protein, this buffered soln conversion system carries out with macromole lyophobic dust (such as cyclodextrin (the cyclodextrin)) solution of concentration from low to high, make by this HOXB4H protein of sex change again folding revert to natural form (native form).
In still another embodiment of the process, the TAT-HOXB4H protein of purifying can be stored in IMDM (HyClone) substratum (store buffer solution 1), 4 DEG C or-20 DEG C.
In still another embodiment of the process, the TAT-HOXB4H protein of purifying can be stored in DMEM (HyClone) substratum (store buffer solution 2), 4 DEG C or-20 DEG C.
In still another embodiment of the process, the histidine mark of TAT-HOXB4H protein C end can remove in the reach carrying out vivo medicine-feeding.
In still another embodiment of the process, the histidine mark of TAT-HOXB4H protein N terminal can remove in the reach carrying out vivo medicine-feeding.
In still another embodiment of the process, TAT-HOXB4H protein N terminal and C end histidine mark can remove in the reach carrying out vivo medicine-feeding.
C. medical constituent is prepared
When with pharmaceutically acceptable supporting agent in conjunction with time, TAT-HOXB4H can be used as medical constituent use.Except TAT-HOXB4H protein and supporting agent, this medical constituent can comprise other known material in thinner miscellaneous, weighting agent, salt, buffer reagent, tranquilizer, solubility promoter and this area." pharmaceutically acceptable " refers to can not the non-toxic material of the bioactive validity of interferon activity agent.The characteristic of supporting agent is depending on route of administration.
Because dosage is homogeneous and easy administration, it is more favourable constituent to be filled a prescription into dosage unit form system.Dosage unit form refers to herein, for being treated the physically discerptible unit of object unit dosage.Each unit contains the promoting agent of a predetermined amount, in order in conjunction with desired medical carrier, produces desired result for the treatment of.The specification system of dosage unit form of the present invention is depending on the peculiar property of promoting agent, the particular treatment effect that will reach.
Typical administration routes, comprises in oral, external application, non-bowel (such as sublingual or suck), sublingual, rectum, vagina and nose." non-bowel (parenteral) " to comprise in (intrathecal) in subcutaneous (subcutaneous), (intracutaneous), intravenous (intravenous), the intramuscular injection (intramuscular) of intracutaneous, intrasternal (intrasternal), intracavernous injection, sheath, duct (intraurethral) injection and any suitable implantttion technique in (intrameata), urethra herein.Medical treatment constituent assignment side becomes to allow that the promoting agent contained by it has bioavailability when giving patient.Medical treatment constituent can give patient by one or more dosage device, and such as a lozenge can be single dose unit, and can contain a plurality of dosage device with the container of spray form.
When with oral give to treat the TAT-HOXB4H protein of significant quantity time, bonding agent can be with the form of lozenge, capsule, medicinal powder, solution or elixir (elixir).When with lozenge form administration, medical constituent of the present invention separately can comprise solid carriers such as gelatin, or adjuvant.Lozenge, capsule, medicinal powder containing the bonding agent (binding agent) of 5% to 95%, can be preferably the bonding agent containing 25% to 90%.Also developer or seasonings can be added.Film clothing layer can be used.When administration in liquid form, liquid carrier such as water, oil (petroleum), animal oil or vegetables oil (such as peanut oil, mineral oil, soybean oil, sesame oil) or synthetic oil can be added.Medical constituent in liquid form separately can comprise normal saline solution, glucose or other carbohydrate solution or glycol such as ethylene glycol, propylene glycol or polyoxyethylene glycol.When administration in liquid form, medical constituent containing the bonding agent of 0.5% to 90%, can be preferably the bonding agent containing 1% to 50%.
When giving the TAT-HOXB4H protein of a treatment significant quantity with vein, skin or subcutaneous injection, it can be the form of non-bowel (parenterally) the acceptable aqueous solution without pyrogeneous substance.Preparation have due pH value, isotonicity, stability non-bowel can accept protein soln, belong to the known technology of this technical field.In certain embodiments, the Zhang Zaiti such as can to comprise for vein, skin or hypodermic medical constituent, such as: sodium chloride injection, ringer's solution, glucose injection, glucose and sodium chloride injection, lactated Ringer's injection liquid or other carrier well known in the art.Medical constituent of the present invention also can comprise tranquilizer, sanitas, buffer reagent, antioxidant or other additive well known in the art.
When carrying out subject treatment method or using of the present invention, first giving user, a such as mammal by the TAT-HOXB4H protein for the treatment of significant quantity, certainly, also can be the mankind." treatment significant quantity " refers to that the total amount of promoting agent in this medical constituent is enough to produce positive effect herein, and such as symptom alleviates, cure or increase curative ratio.When be used in single-activity agent and individually dosed time, " treatment significant quantity " singly refers to this promoting agent.When for a combination, " treatment significant quantity " is total refer to that all promoting agents are enough to produce the resultant of result for the treatment of, no matter those promoting agents refer to serial combination medicine-feeding or combination medicine-feeding simultaneously.
The seriousness of the content of TAT-HOXB4H protein in the present invention's medical treatment constituent depending on wish treatment symptom and the Age and sex of the treatment essence essential, patient had previously accepted and patient.Finally, attending doctor can determine few patients's active agent delivery amount.Just start, attending doctor can give the promoting agent of low dosage, and observes reaction.The promoting agent of larger dose can be given until patient obtains suitable result for the treatment of, now generally can not increase dosage again.Medical constituent in order to implement the inventive method can be about the TAT-HOXB4H protein of 1g to about 1mg containing per kilogram of body weight.The dosage range giving user can be selected from 1 μ g/kg to 1mg/kg, 1 μ g/kg to 0.5mg/kg, 1 μ g/kg to 0.1mg/kg, 10 μ g/kg to 0.5mg/kg, 10 μ g/kg to 0.1mg/kg, 100 μ g to 0.5mg/kg, 250 μ g/kg to 0.5mg/kg.In addition, dosage range can be selected from 50 μ g to 100mg, 100 μ g to 50mg, 500 μ g to 50mg, 1mg to 50mg.Use the disease severity of intravenous injection time depending on wish treatment and the specific reaction of few patients of the present invention's medical treatment constituent.In still another embodiment of the process, the time imposing TAT-HOXB4H protein of the present invention can be the continuous intravenous injection of 12 to 24 hours.In still another embodiment of the process, the sustainable use of TAT-HOXB4H protein of the present invention, as long as patient continues carrying out chemotherapy or radiation cure.TAT-HOXB4H protein can intravenous injection 10-100 μ g/kg, one day twice, 4.5 to 5 days.Seance circulation may just be enough to amplifying candidate stem cell in vivo.Finally, attending doctor can determine the suitable intravenous injection time using the present invention's medical treatment constituent.
Toxicity of compound and Curative effects can the pharmaceutics program of standard determine with cell cultures or laboratory animal, such as LD
50(total 50% lethal dose) and ED
50(total 50% dose therapeutically effective).Toxicity and treatment between dose ratio be therapeutic index, it can LD
50/ ED
50represent.The data of cell cultures and laboratory animal can be used for assessing the dosage range for the mankind.Compound dosage can comprise ED
50and within the scope of the little or nontoxic circulation composition of toxicity.Depending on used formulation and route of administration, dosage can change in above-mentioned scope.The dose therapeutically effective of TAT-HOXB4H can by cell culture test according to a preliminary estimate.In animal model, dosage can be reach to comprise IC
50the circulating plasma concentration range of (also namely test protein reaches the concentration of maximum symptom suppression half), just as the mode that cell cultures determines.Plasma concentration can utilize high performance liquid chromatography to measure.The effect of any given dose can be monitored by suitable biologic assay (Bioassay).The biologic assay be applicable to include, but are not limited to, and utilizes the CD34 being marked with fluorescent probe (such as FITC)
+stem cell antibody measures the CD34 in monocyte
+stem cell, and utilize flow cytometer to measure the LY5 cell proportion in Peripheral blood or marrow hemopoietic stem cells.Polynucleotide of the present invention and protein system expect can represent following one or more purposes or biological activity.The purposes of present protein or activity or can use the mode of this protein to provide by administration, also or the mode of the polynucleotide of this protein of coding (such as gene therapy or be applicable to send into the carrier of DNA) can be used to provide by administration.
III. hematopoietic stimulation method in body
A. the patient treated is needed
Medical constituent of the present invention can be used for treating autoimmune disorders, disease of immune deficiency and hematologic disease.In addition, medical constituent of the present invention can be used for the time of recovery after improving hematopoietic stem cell transplantation.Medical constituent of the present invention can be used for treating the patient suffering from lymphoma, leukemia, hodgkin's disease and myeloproliferative disease.In addition, hemopoietic stem cell lacks and the congenital disorders that cause and hypoplasia anaemia also can medical constituent treatments of the present invention.
In addition, medical constituent of the present invention can be used for hemopoietic stem cell contributor and particle glomus cell somatomedin (G-CSF) insensitive patient.
In still another embodiment of the process, TAT-HOXB4H is the sole active being used to mobilizing hematopoietic stem cells, and Ro 2-9757 (5-FU) does not give contributor, no matter be as pre-treatment or complex therapy plan.
Can the carrying out of malignant tumour and relative disease be comprised by the extra disease of the present invention's medical treatment constituent treatment or the symptom relevant to increasing cells survival and/or shift, such as promyelocytic leukemia (comprises acute leukemia (such as: acute lymphoblastic leukemia, acute myelocytic leukemia (comprises myelocyte, promyelocyte, granulocyte, monocyte, erythroleukemia)) and chronic leukemia (such as: chronic granulocyte (granulosa cell) leukemia and chronic lymphocytic leukemia), marrow forms abnormality disease (myelodysplastic syndrome), true property erythrocytosis (polycythemia vera), lymphoma (such as: hodgkin's disease and non-Hodgkins disease), multiple myeloma,
macroglobulinemia and solid tumor, comprise sarcoma and cancer knurl, such as: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliocyte sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovioma, mesothelioma, Ewing' s tumor, leiomyosarcoma, rhabdosarcoma, colorectal carcinoma, cancer of pancreas, mammary cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, rodent cancer, gland cancer, syringocarcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cancer of bile ducts, suede cancer, spermatocyte knurl, embryoma, prestige Mu Shi knurl, cervical cancer cancer, tumor of testis, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pine nut adenoma, hemangioblastoma, acoustic tumor, glioma, melanoma, neuroblastoma and retinoblastoma.
The present invention is not limited to specific method, experimental plan, cell strain, animal species or genus or following reagent.Be used for describing the term of specific embodiment can not in order to limit scope of the present invention, scope of the present invention should be as the criterion with the scope of claims.Used herein, " " and " being somebody's turn to do " of singulative, unless outside Wen Yi refers else, also comprised a plurality of.Therefore, for example, " cell " refers to one or more cell, and comprises equipollent known in the art.
Unless otherwise defined, otherwise all technology and Institute of Science vocabulary have the understanding of connotation identical with being familiar with this operator in the technical field of the invention.Although any method, assembly is similar with material or be equal to described here, and all can be used for practice or test the present invention, preferred method, assembly and material are in present description.All publications of herein mentioning and patent are incorporated in order to describe and open at this, for example, are described in the structure in publication and method, can be combined with invention described at present.Disclosing before the publication previously and herein discussed only is provided as this case expiration date.All neither may be interpreted as admits that contriver cannot rely on prior inventions open to predict this.
Definition:
" stem cell " is a kind of multipotency or multipotential cell, its capable self, to keep not breaking up, and becomes and breaks up.Stem cell, at least exists in life naturally animal, can divide without limit.Stem cell is not terminal differentiation, and namely they are not at differentiation pathway end.Work as stem cell division, each daughter cell, a stem cell can be remained and maybe can move towards out the road that causes terminal differentiation." mosaic " stem cell refers to that a part of DNA of stem cell belongs to a heterologous organism.
" hematopoiesis " cell refers to a kind of cell relating to hematopoiesis (also namely precursor cell forms the process of ripe blood cell).Adult, hematopoiesis occurs in marrow.Early stage in growth, in different developmental phases, hematopoiesis occurs in different places; Original blood cell comes across yolk sac, and afterwards, blood cell was formed in liver, spleen and marrow.Hematopoiesis has complicated regulation and control, comprises hormone (such as erythropoietin, somatomedin (such as G CFS and cytokine (be such as situated between white element))).
" carrier " used herein refers to the nucleic acid molecule that can transport another kind of nucleic acid, and this another kind of nucleic acid is connected on this nucleic acid molecule.The carrier of one type is one " plastid ", and this refers to a circular distrand DNA ring, and extra DNA fragmentation can be bonded on wherein.The carrier of another kind of type is virus vector, and wherein extra DNA fragmentation can be bonded in viral genome.Some carrier is had the ability self-replicating (such as having bacteria carrier and plasmid episomal (episomal) mammalian vector that a bacterium copies source) in sent into host cell.Other carrier (such as non-add physique grain (episomal) mammalian vector) can be incorporated in the genosome of host cell when sending into host cell, thus copies along with host genome.In addition, some carrier is its gene expression connected of commander of having the ability.This carrier is be called " restructuring display carriers " (or briefly " display carriers ") at this.In general, for the display carriers of DNA recombinant technology normally with the form of plastid.In the present invention, " plastid " and " carrier " can exchange use, because plastid is the most frequently used carrier format.But, the present invention includes the display carriers of other form with identical functions, such as virus vector (retrovirus of such as replication defective, adenovirus and adeno-associated virus).
" conversion " used herein refers to and an exogenous polynucleotide is sent into host cell, no matter how to send into: for example, and direct sorption enhanced, transfection and infection etc.Specific transfection method sees hereafter.Exogenous polynucleotide may be with the form of non-insertion vector (such as: plasmid), or may be integrated in host genome.
In general, " protein " refers to that any two or more Individual amino acids is by winning polymkeric substance that peptide bond (peptide bond) engages (no matter whether naturally-occurring), this victory peptide bond refers to when bond is at the carboxyl carbon atom of the carboxylic acid group of the α carbon of an amino acid (or amino-acid residue), with the amino nitrogen atom of bond at the amido of the α carbon of adjacent amino acid (or amino-acid residue), the covalently bonded formed.The atom (being also alpha-carbon atom, carboxyl carbon atom (and its Sauerstoffatom substituting group) and amino nitrogen atom (and its hydrogen atom substituent)) that this victory peptide bond connects and comprises forms protein " winning peptide backbone " more.In addition, " protein " used herein can be understood as and comprises " winning peptide " and " victory peptide " (it can use sometimes alternately) more.Equally, protein fragments, analogue, derivative and mutation-ure referred to here as " proteins ", except as otherwise noted, also can should be considered to be one " protein ".Protein " fragment " refers to the many victory peptide comprising and be less than an all amino-acid residue of protein.Be understandable that, protein " fragment " may be that one is held at N, C holds or the protein of inside (such as caused by natural montage (splicing)) brachymemma, can be mutation-ure and/or derivative.Protein " mac function (domain) " is also a fragment, and it comprises the amino-acid residue given needed for protein biochemistry activity, corresponding to the protein naturally existed.
" restructuring " refers to a nucleic acid molecule herein, genome, gene, virus, semi-synthetic, or synthesis, its part or all polynucleotide non-natural." restructuring ", for a protein or many victory peptides, refers to the many victory peptide produced with recombination of polynucleotide performance.
One " isolated ", " purifying ", " isolated in fact " or " in fact purifying " molecule (as won peptide or Nucleotide one) refers to be operated manually more makes compared to naturally there being higher concentration.For example, when target protein is by isolated, purifying, isolated in fact or purifying in fact, refer to have 50% at least, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the naturally occurring nonstandard proteinaceous substances of 98%, 99% or more is removed." isolated " used herein, " purifying ", " isolated in fact " or " in fact purifying " molecule comprise recombinant molecule.
SCID " mouse " refers to severe combined immunodeficient (SCID) mouse model, and SCID can cause the major defect of developing immune system.No matter be T or B lymph corpuscle, these mouse are neither sufficient or lack completely.This SCID suddenlys change and seems to damage the restructuring of antigen receptor gene, causes lacking functional T and B lymph corpuscle.Other hematopoetic cell types can normal development and running.SCID mouse supports that normal lymphoid ball breaks up at any time, and can recombinate with the normal lymphoid ball from homogenic or Allogeneic mouse or recombinate with human lymphocytes's ball.These mouse also support the growth of recessive allele and heterogenous gene tumour.Therefore, SCID mouse allows the diffusion of some human tumors to increase, particularly disease in the blood system and malignant melanoma, therefore can be used for studying malignant tumour.
" user ", " individual ", " host " and " patient " are used interchangeably herein, in order to refer to the animal lived, comprise the mankind and inhuman animal.User may be, for example, an organism having an immunocyte, this immunocyte source of resisting stimulates and produces reaction, and the stimulation can transmitted the receptors bind via cell surface and suppress signal to produce to react.User can be a mammal, such as people or inhuman Mammals, such as dog, cat, pig, ox, sheep, goat, horse, rat and mouse." user " does not get rid of, relative to disease or each side, and completely normal individual.
" treatment " refers to a therapeutic or preventative measure.Treatment can put on the user with medical condition or finally can obtain disease patient, in order to prevention, treatment, the symptom of disease incuring loss through delay, reduce seriousness or improve one or more diseases or repeatedly occur, or it exceedes the life span not have desired by treatment in order to extend user's chien shih lifetime.
" treatment significant quantity " refers to that main compound can cause the amount of anticipation reaction (biology of the tissue such as assert via researcher, animal doctor, doctor or other clinicist, system, animal, animals or humans or medical response).
Example concrete is below, to be regarded as being only illustrative, is no matter neither limit other parts of the present invention in any situation.Those of ordinary skill can implement the present invention based on describing of present specification in the art.
Constructing of example example 1:pET21b-His-TAT-HOXB4-His plastid
A () N-holds and C-end wins the amendment of the pET21b plastid of peptide containing histidine mark and TAT signal
The pET21b display carriers of N-end containing histidine mark and TAT signal victory peptide obtains by inserting following oligonucleotide at pET21b plastid:
5 '-TATGCACCACCACCACCACCACTACGGCCGCAAGAAACGCCGCCAGCGCCGCCGGC G-3 ' (underlying stock (sense)) and
5 '-CTAGCGGCGCTGGCGGCGTTTCTTGCGGCC GTAGTGGTGGTGGTGGTGGTGCA-3 ' (anti-stock (antisense)).C-holds histidine mark to be originally just present in pET21b plastid.
B HOXB4 choosing is grown and is showed plastid into amended pET21b by ()
DNA fragmentation containing HOXB4 open reading frame (ORF) and extra six histidine encoding sequences, with MGC54130 plastid (GeneDiscovery, Taipei, Taiwan.Cat.No.5533346) be template, utilize polymerizing enzyme chain reaction (PCR) amplify and obtain, (subclone) is grown in time choosing of the HOXB4cDNA fragment of PCR gained and shows plastid to amended pET21b.The plastid constructed and nucleotide sequence system are as shown in Figures 2 and 3.
Example example 2: at intestinal bacteria performance TAT-HOXB4H recombinant protein
PET21b-His-TAT-HOXB4-His is showed plastid transformation to BL21 (DE3) pLysS (Novagen) e. coli strains.Make the cell grow overnight at 37 DEG C through transforming.Overnight culture is diluted to initial OD
600value is 0.05, and grows to OD at being placed in 37 DEG C
600value is 0.5, then at 37 DEG C, carries out induction performance 3 hours with 1mM isopropylthio-β-D-semi-lactosi (IPTG), and period is with acutely rocking.
Example example 3:TAT-HOXB4H purifying recombinant proteins
After inducing action, by cell with collected by centrifugation and settling flux in buffer A (8M urea, 20mM HEPES, 0.5mM DTT and 100mM NaCl pH8.0).Cell suspending liquid is passed through French press tri-times, and by cell lysates with 20,000 × g makes it clarify in centrifugal 30 minutes at 4 DEG C.Supernatant liquor is adjusted to 10mM imidazo application of sample to HisTrap chelating tubing string (Amersham Pharmacia).Combining albumen with the imidazoles that 50,100 and 250mM are formulated in buffer A carry out molten from.By stream part (fraction) application of sample containing TAT-HOXB4H to there is buffer B (4M urea, 20mM HEPES and 50mM NaCl pH6.5) MonoSP tubing string, with 1.5M NaCl and 20mM HEPES (pH8.0) molten from.
The renaturation (renaturation) of example example 4:TAT-HOXB4H recombinant protein
By molten from the TAT-HOXB4H proteolytic in stream part and sex change in one containing the solution of sex change salt (such as guanidine hydrochloride (guanidine hydrochloride)), then it is mixed with D-PBS-T buffered soln (the twice phosphate buffer soln containing 0.1%Triton X-100).The ratio of TAT-HOXB4H protein soln and D-PBS-T buffered soln is 1:4.Formed mixed solution is added the 10K protein concentration tubing string (centricon tube) (50ml or 15ml) with water (10ml or 3ml) pre-treatment, centrifugal 3000rpm, 10 minutes.In this step, sex change salt system is by D-PBS-T buffer exchange, and the Triton X-100 system in D-PBS-T damping fluid can be combined with the hydrophobicity of HOXB4H protein (Hydrophobic) region
Then filter or buffered soln displacement step 10 times is performed for more than with 10K protein concentration tubing string (centricon tube), with 1000-2500g/min centrifugal speed, be sequentially the IMDM store buffer liquid containing 1mM, 2mM, 3mM, 4mM and 5mM beta-cyclodextrin (beta-cyclodextrin) by buffer exchange, wherein replace centrifugal twice with often kind of concentration under each centrifugal speed.Collect the sample retained in concentrated tubing string, and be stored in-20 DEG C of refrigerators.
After purifying, the homogeneity system of TAT-HOXB4H protein is with SDS-polyacrylamide gel and coomassie staining analysis.As shown in Figure 5, through the about 3-5 of gain in yield times of the TAT-HOXB4H more original TAT-HOXB4 protein of HisTrap and MonoSP purifying.PTAT-HA-HOXB4 plastid is that lid savart lattice (Guy Sauvageau) doctor of Canadian Montreal university (University of Montreal) given.PTAT-HA-HOXB4 plastome is converted into BL21 (DE3) pLysS (Novagen), and TAT-HOXB4 protein purification system is as described in the people such as Krosl (2003).
The stability of example example 5:TAT-HOXB4H recombinant protein
The stability system of TAT-HOXB4H is with SDS-polyacrylamide gel analysis.During storage, the TAT-HOXB4H of total length may be degraded to 30kD and 10kD fragment.As shown in Figure 6, even if TAT-HOXB4H protein of the present invention is at PBS, stores in-4 DEG C and still stabilize for 16 hours.
In addition ,-4 DEG C and-20 DEG C are stored in, the TAT-HOXB4H of PBS and IMDM damping fluid, with 10%SDS-polyacrylamide gel electrophoresis and coomassie staining analysis.As shown in Figure 7, when being stored in IMDM store buffer liquid, TAT-HOXB4H protein of the present invention can maintain for 4 weeks.
Example example 6:TAT-HOXB4H recombinant protein affects for the hematopoiesis of Balb/c mouse
TAT-HOXB4H recombinant protein utilizes Balb/c mice study for hemopoietic stem cell by bone marrow mobilization to the system that may affect of Peripheral blood.Every day, four subcutaneous injections gave mouse TAT-HOXB4H recombinant protein (in PBS) for 4 days.For understanding dose response, the dosage of test group (n=21) is 1 μ g, 5 μ g, 10 μ g, 15 μ g ... to 100 μ g/kg body weight.One group of control group injection PBS, the G-CSF of another group control group then subcutaneous injection 4 days 5 μ g/kg body weight twice daily.
Get each group of periphery blood and carry out flow cytometry analysis, can CD34 be obtained
+the ratio of stem cell in monocyte (MNC).Result lists in table one with the form of mean+SD.
Table one
| Group | TAT-HOXB4H(g/kg) | CD34 +/MNC(％) |
| 1 | 1 | 0±0.03 |
| 2 | 5 | 0.3±0.05 |
| 3 | 10 | 0.45±0.03 |
| 4 | 15 | 0.42±0.01 |
| 5 | 20 | 0.38±0.05 |
| 6 | 25 | 0.41±0.02 |
| 7 | 30 | 0.35±0.21 |
| 8 | 35 | 0.33±0.11 |
| 9 | 40 | 0.29±0.16 |
| 10 | 45 | 0.46±0.01 |
| 11 | 50 | 0.45±0.02 |
| 12 | 55 | 0.42±0.06 |
| 13 | 60 | 0.44±0.02 |
| 14 | 65 | 0.41±0.04 |
| 15 | 70 | 0.41±0.03 |
| 16 | 75 | 0.49±0.01 |
| 17 | 80 | 0.45±0.04 |
| Group | TAT-HOXB4H(g/kg) | CD34 +/MNC(％) |
| 18 | 85 | 0.46±0.07 |
| 19 | 90 | 0.44±0.02 |
| 20 | 95 | 0.41±0.01 |
| 21 | 100 | 0.42±0.05 |
| Control group (PBS) | 0 | 0.002 |
| Control group (G-CSF) | 0 | 0.5±0.03 |
CD34 in periphery blood
+the ratio series of/MNC is in table one.3-21 experimental group (dosage is at least more than the 10 μ g/kg body weight) display accepting TAT-HOXB4H is similar to the mobilization effect of injection G-CSF control group.
Get the marrow of the marrow of experimental group 3 (TAT-HOXB4H dosage is 10 μ g/kg body weight) mouse and the control group mice of injection G-CSF and PBS, with CD34
+fITC-conjugated antibody (Becton Dickinson) phenotype, and with flow cytometry analysis.The mouse bone marrow cells (Fig. 8 C) of injection TAT-HOXB4H, its CD34
+stem cell is more than the mouse bone marrow cells of injection G-CSF and PBS.These results display injection TAT-HOXB4H recombinant protein can increase the hemopoietic stem cell in mouse bone marrow cells and in Peripheral blood simultaneously.
Example example 7:TAT-HOXB4H recombinant protein affects for the hematopoiesis of rhesus monkey
TAT-HOXB4H recombinant protein utilizes the public rhesus monkey research of growing up in the effect system of monkey class.Test group I (n=5) is that four intravenous injections every day give TAT-HOXB4H recombinant protein (dosage 10 μ g/kg body weight) for 4 days.Test group II (n=5) is that four intravenous injections every day give TAT-HOXB4H recombinant protein (dosage 10 μ g/kg body weight) and subcutaneous injection G-CSF (dosage 5 μ g/kg body weight) for 4 days.Control group I injection PBS, control Group II then subcutaneous injection 4 days G-CSF (dosage 5 μ g/kg body weight) twice daily.The periphery blood getting all monkeys carries out flow cytometry analysis, can obtain CD34
+the ratio of stem cell in monocyte (MNC).The results are shown in table two.
Table two
As shown in Table 2, the monkey (experimental group I) only accepting TAT-HOXB4H demonstrates better mobilizes effect than the monkey (control Group II) of injection G-CSF.The monkey (experimental group II) of simultaneously injecting TAT-HOXB4H and G-CSF demonstrates more effective than the mobilization of the monkey (control Group II) of injection G-CSF.
Get the monkey sample of bone marrow of injection TAT-HOXB4H, G-CSF and PBS, with CD34
+fITC-conjugated antibody (Becton Dickinson) phenotype, and with flow cytometry analysis.The monkey marrow (Fig. 9 C) of injection TAT-HOXB4H, its CD34
+stem cell line is far more than the CD34 in the monkey marrow (Fig. 9 D) of the monkey marrow (Fig. 9 A) of injection G-CSF, the monkey marrow (Fig. 9 B) injecting TAT-HOXB4H+G-CSF and injection PBS
+stem cell.
Example example 8:TAT-HOXB4H recombinant protein replys impact for the hematopoiesis of NOD-SCID mouse
By 10
4lin
-/ CD34
+cell and through 10 of radiation exposure
5cD34
-helper is injected in NOD-LtSz-scid/scid (NOD-SCID) mouse (through radiation exposure (2.5Gy)).Above-mentioned mouse is divided into two groups at random, and one group of (n=28) intravenous injection every day, twice TAT-HOXB4H protein (dosage 10 μ g/kg body weight), another group (n=28) every day injects twice PBS.After implantation, with mankind CD45
+the cell ratio existed in muroid periphery blood reaches the mouse number of more than 0.1% to assess hematopoiesis reply.As shown in Figure 10, the mouse of injecting TAT-HOXB4H recombinant protein can be observed preferably hematopoiesis and replys.
Example example 9:TAT-HOXB4H recombinant protein replys impact for the hematopoiesis of Balb/c mouse after accepting Cisplatin chemotherapy
Repeat with cisplatin intravenous injection five weeks large Balb/c mouse, until Ly5 (murine CD45) cell number reduces to 10% of original number in its Peripheral blood.The mouse of injecting cisplatin is divided into two groups at random, and one group of (n=28) intravenous injection every day, twice TAT-HOXB4H protein (dosage 10 μ g/kg body weight), another group (n=28) every day injects twice PBS.With the Ly5 cell existed in the flow cytometer periodically all mouse Peripheral blood of measure analysis.Hematopoiesis is replied ratio system and is assessed with the ratio of the Ly5 cell number in Peripheral blood relative to original number.As shown in figure 11, the mouse of injecting TAT-HOXB4H recombinant protein can be observed preferably hematopoiesis and replys.
The animal models used in these experiments are confirmed to be in the art can in order to the result of predict human patient, such as: the people such as Broxmeyer (2005) The Journal of ExperimentalMedicine, 201,1307-1318; The people such as Larochelle (2006) Blood 107,3772-3778.
The above; be only the specific embodiment of the present invention, but protection scope of the present invention is not limited to this, is anyly familiar with those skilled in the art in technical scope of the present invention; change can be expected easily or replace, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claim.
Claims (8)
1. the preparation method of a TATHOXB4H recombinant protein, for the preparation of in vivo offeing medicine, the composition of described recombinant protein is promoting that hemopoietic stem cell is applied to the medicine of periphery blood by bone marrow mobilization, it is characterized in that: described composition at least comprises the TAT-HOXB4H recombinant protein of a significant quantity, the amount of described protein is enough to the absolute number of hemopoietic stem cell in increase by user's marrow in need, thus promote that hematopoietic stem cell mobilization is to the periphery blood of described user, wherein said TAT-HOXB4H recombinant protein is after escherichia coli host performance, purifying is carried out: i) the impure solution of this protein of performance is passed into a HisTrap tubing string with following method, (ii) this HisTrap tubing string is rinsed, (iii) this partially purified protein is molten from going out by this HisTrap tubing string, and form the protein soln of a part of purifying, (iv) this partially purified protein soln is passed into a MonoSP tubing string, v () rinses this MonoSP tubing string, (vi) by the protein of purifying, in denatured form, molten from going out by this MonoSP tubing string, and utilizing hydrophobic compound by the molten denatured protein from going out with the following step folding again: this molten denatured protein from going out and a hydrophobic compound solution are mixed to form a protein and hydrophobic compound solution by (i), (ii) this protein and hydrophobic compound solution are desalted and a protein and hydrophobic compound desalt solution, (iii) utilize ultra-filtration processing procedure to be desalted in solution by this protein and hydrophobic compound by this hydrophobic compound to remove, and this protein is stored in IMDM substratum or DMEM substratum after purifying, to improve the stability of protein purification.
2. preparation method according to claim 1, is characterized in that: described user in need is a hemopoietic stem cell contributor.
3. preparation method according to claim 1, is characterized in that: described user in need is the patient carrying out autologous hematopoietic stem cell transplantation.
4. preparation method according to claim 1, is characterized in that: described user in need is an insensitive patient of particle glomus cell somatomedin.
5. preparation method according to claim 4, is characterized in that: the disease that described user system suffers from congenital hemopoietic stem cell shortage and causes.
6. preparation method according to claim 5, is characterized in that: described user system suffers from congenital hypoplasia anaemia.
7. preparation method according to claim 1, is characterized in that: described user system suffers from hematologic disease, solid tumor or Immunological diseases.
8. preparation method according to claim 7, is characterized in that: described hematologic disease system is selected from the group be made up of lymphoma, leukemia, hodgkin's disease and myeloproliferative disease.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111434694A (en) * | 2019-01-11 | 2020-07-21 | 台湾尖端先进生技医药股份有限公司 | N-terminal labeled histidine recombinant TAT-HOX family protein and preparation method thereof |
| CN111620938A (en) * | 2019-02-27 | 2020-09-04 | 台湾尖端先进生技医药股份有限公司 | Mutant HOXB4 recombinant protein and preparation method and application thereof |
| CN111620938B (en) * | 2019-02-27 | 2022-05-03 | 台湾尖端先进生技医药股份有限公司 | Mutant HOXB4 recombinant protein and preparation method and application thereof |
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